Glycomics has emerged with proteomics as an area for development and exploration in the postgenomics era (Blixt et al (2004)). Despite the increasing awareness of the biological significance of carbohydrates, the study of carbohydrate-protein interactions still encounters much difficulty. There is a need for the development of highly sensitive and high-throughput methods for identification and binding study of carbohydrates recognized by various receptors (Chung-Yi et al (2009)).
The immobilization of glycans on the derivatised surface of substrates is a commonly employed method of fabricating glycan microarrays. Blixt et al (2004) discloses immobilisation of amine functionalised synthetic glycan ligands on N-hydroxysuccinimide (NHS) activated glass slides using a custom made robotic printing arrayer. Bovin and Huflejt (2008) have reviewed the use of binding chemistries exploiting amide bond formation. Short spacers are used to reduce non-specific contacts to a minimum. Attachment to a flexible layer of polyethylene glycol on a glass surface is presented as assuring availability of glycan moieties for interaction with binding molecules.
The localization of glycans to the surface of substrates in the form of neoglycolipids has also been employed as a method of fabricating glycan microarrays. Chai et al (2003) describe a multiwell-binding assay in which neoglycolipids are diluted either in methanol, or in methanol containing the carrier lipids egg lecithin and cholesterol. The dispersions of neoglycolipids are then used to coat the wells of the multiwell plates. Chai et al (2004) describe the bandwise application of the dispersions of neoglycolipids by a spray-on technique employing a sample applicator comprising a single syringe as applicator (LINOMAT IV, Camag, Switzerland)
Fukui et al (2005) and Huang et al (2006a, 2006b) have each described a non-covalent glycoarray assembly method utilising lipid-linked saccharides and oligosaccharides. Both methods employ reductive amination to produce a lipid-linked saccharide of oligosaccharide (neoglycolipid). In the method of Fukui et al (2005) oligosaccharides were conjugated to 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) directly or after mild periodate oxidation. The neoglycolipids were then applied by jet spray as bands or spots onto nitrocellulose membranes. In the method of Huang et al (2006a, 2006b) the reaction to produce lipid-linked saccharides uses an excess of saccharides in order to exhaust the tetradecylamine employed in the reaction. The lipid-linked saccharides were then applied to multi-well high binding polystyrene plates.
Liu et al (2006) describe the preparation of neoglycolipids from N-aminooxyacetal DHPE (AOPE) by a chemoselective oxime-ligation reaction with reducing sugars. The binding of the neoglycolipids by antibodies and lectins was assayed by an enzyme-linked immunosorbent assay (ELISA) in plastic microwells as described by Chai et al (2003). In these studies the neoglycolipids were incorporated into liposomes for arraying and spotted onto nitrocellulose membranes or robotically arrayed onto nitrocellulose-coated glass slides.
Palma et al (2006) and Campanero-Rhodes et al (2007) describe the preparation of arrays of natural and synthetic glycolipids and neoglycoplipids by printing on nitrocellulose-coated glass slides using a non-contact piezoelectric arrayer (PIEZORRAY, Perkin-Elmer, United Kingdom). Liu et al (2007) also describes the use of this non-contact piezoelectric arrayer. The arrayer employs an assembly containing four PIEZOTIP™ dispensers to dispense sub-nanoliter to nanoliter volumes with 20 to 25 μm accuracy and precision.
It is an object of the invention to provide an improved method for the localisation of functional moieties, including glycans, to the surface of substrates.
It is an object of the invention to provide a method of fabricating diagnostic test cards and sticks, microarrays and multiwell plates.
It is an object of the invention to provide templates for use in the fabrication of diagnostic test cards and sticks, microarrays and multiwell plates microarray formats by the method that improve accuracy and reliability of assay results.
These objects are each to be read disjunctively with the object to at least provide the public with a useful choice.